TW200401826A - Method for isolating and measuring proliferation of long-term label retaining cells and stem cells - Google Patents

Abstract

This invention relates to a method for separating long-term label retaining cells or stem cells. In particular, this invention relates to a method for separating long-term label retaining cells and/or stem cells from tissues or individuals and for measuring proliferation rates of long-term label retaining cells and stem cells, as well as determining clonal expansion (proliferative history) of cell lineages from the tissues of the individual. The cells may be double-labeled with a cell-lineage marking label and isotopically labeled DNA synthesis precursor prior to physical separation.

Description

200401826 发明. Description of the invention: 袓-Patent information and related participants. This application claims the priority of US Provisional Patent Application No. 60 / 370,599 filed on April 5, 2002. [Technical Field to which the Invention belongs] The present invention relates to a method for separating long-term labeled retentive cells and / or stem cells from unlabeled retentive cells and / or non-stem cells. In particular, the present invention relates to a method for isolating stem cells from tissues or individuals, a method for determining the proliferation rate of long-term labeled retained cells and / or stem cells, and a method for identifying homologous proliferation (history of proliferation) of tissue and individual cell lineages. [Prior art] The number of cell divisions performed by a specific cell lineage is the focus of many diseases and normal physiological processes. Cell division within a specific lineage, also known as the history of homologous proliferation or proliferation of specific cell lines (especially stem cells), affects the risk of cancer (i.e., canceration), the rate at which fixed DNA damage becomes a permanent mutation (i.e., mutation, causing Domain secretion, canceration, evolution rate), the cell's response to antigenic stimulation (ie vaccine efficacy), spermatogenesis (ie male fertility), fat regeneration from pre-adipocytes (ie, epiphyseal month purpose naturally tired) ), The total number of epithelial cells (i.e., tissue homeostasis), neurogenesis in the brain, and other medical conditions and diseases. Stem cells (both embryonic and adult) have received widespread attention in many biological and medical fields, including normal tissue homeostasis, the pathogenesis of disease, and therapeutics. Epithelial cells line the hollow tubes (lumens) on the outside of the body and in contact with the outside world. Epithelial tissue includes skin, gastrointestinal tract (such as colon, small = stomach, esophagus, mouth, exocrine glands, etc.), urogenital tract (such as bladder, pre-84752 200401826 gonad, ovary, uterus, cervix, vagina, urethra) , Hepatobiliary tract (liver, gall bladder and gallbladder), breast and respiratory system (such as bronchus, trachea, alveoli, nasopharynx, etc.). These tissues are characterized by continuous cell renewal. Older cells shed from the surface of the tissue to the inner cavity or the outside and are replaced by recently divided and differentiated epithelial cells. The tissue that this cell renewal process is common to all epithelial tissues appears to be epithelial stem cells ( Or mature stem cells). These epithelial stem cells have two central characteristics: first, the unique ability of asymmetric cell division (where one daughter cell produces a cell clone of differentiated cells and the other daughter cell maintains undifferentiated stem cells); and second, slow Cell division speed. I believe that in various functional units of epithelial tissues, such as colonic crypts or breast acinars, there are a small number of stem cells or possible stem cells to maintain the flow of differentiated cells and maintain tissue quality and function in the face of continuous cell loss. . Unfortunately, most stem cells have not yet identified a decisive marker, either on the cell surface or within the cell. Difficulty in identifying the “stem cell” logo Probability may be the basis: Stem cells are less differentiated than the daughter cells that surround them in tissues and are known for their non-expression (such as genes and proteins). Because it is more difficult to isolate cells based on non-emerging rather than on-emerging, stem cells remain difficult to physically isolate. The inability to isolate stem cells hinders the progress of treatments (such as growing stem cells in vitro for the therapeutic regeneration of tissues) and diagnostics (such as cancer risk based on mutation rates or a history of homologous hyperplasia). Stem cells are characterized by longevity. Many longevity-supposed stem cells are documented in the literature in many epithelial tissues (such as skin and small intestine) and mesenchymal tissues (such as bone marrow). After administration of radioactive DNA precursors (such as 3H-thymidine), these long-term labeling-retaining cells (LRCs) 84752 200401826 are long-term retention labels in their nucleus, and in these tissues Most of the cells present in it were unlabeled within a few days because of rapid replacement of differentiated epithelial cells. Several pieces of evidence support the hypothesis that these long-term labeled retention cells (LRCs, ___ hbe / = etaining cells) are indeed the target cell population of carcinogens, the most important of which is the hypothesis that stem cells (or stem cells are highly enriched). It was observed that [hidden in a tissue location expected to be an epithelial stem cell (such as the base of the colonic glandular fossa or in the central portion of the proliferative unit in the skin). Hearing occurs as a smaller fraction of the total differentiated cell population (eg, 5_0 cells in the glandular fossa population of 250 cells). Finally, LRCs in the skin also exhibit long-term retention of DNA adducts known to be carcinogens, consistent with direct DNA binding and mutation initiation in these LRCs. However, the retention of nucleotide- or radio-labeled DNA in the subgroups of cells (LRCs) has failed to allow such cells to be isolated or to directly measure how often they divide. Therefore, the genotype or phenotypic characteristics of stem cells have not progressed; stem cells grown or cultured in vitro have not yet achieved therapeutic use; and cancer risk assessment due to the rate of stem cell mutation is not yet feasible. I recently reported a non-radioactive method for measuring cell proliferation in vivo. Neese et al. PNAS 2002; 99: 15345-15350. DNA replication (and thus cell proliferation) was measured based on the addition of deuterium from nitrified water (2H2O) to the deoxyribose (dR) of newly synthesized DNA. This technique has been used to measure the proportion of LRCs present in a population of cells (such as T cells or mammary epidermal cells) after discontinuing the administration of radon. However, labeling methods have not been used to isolate stem cells or measure stem cell proliferation rates and stem cell homogeneous proliferation. It is true that at present, there is no reliable technology for external detection and isolation of LRCs 84752 200401826 or stem cells, or for measuring the straightening rate of LRCs or stem cells in tissues and individuals. Therefore, there is a great need to identify and quantify LRCs and stem cells. method. Furthermore, it is highly desirable to measure proliferation rates in tissues and individuals, as well as homogeneous proliferation of cell lineages (history of proliferation). [Summary of the Invention] In order to meet these needs, the present invention is directed to a method for separating long-term labeled retention cells (LRCs, 10ng-term label_retaining ⑶⑴) and / or stem cells, and measuring the rate of appreciation of LRCs and / or stem cells in tissues or individuals. . In one format, the present invention is directed to a method for isolating LRCs & or stem cells based on their snap action labeling characteristics. In this method, a cell lineage annotation marker is administered to a tissue or individual. The cell lineage annotation marker is administered to the labeled cells, particularly LRCs and / or stem cells, in a sufficient amount and for a sufficient period of time. Cells are labeled with annotation markers so that they are incorporated into the dividing cells at a sufficient concentration to allow external detection and isolation of the dividing cells. Cell lineage annotation tags are incorporated into the cell's DNA. Cell lineage annotation markers can be halogenated deoxyribonucleic acid (dn) such as bromide deoxyuridine (Brdu, bromodeo-xyuridine) or iodide deoxyuridine (idu, iododeoxyuridine) O Cell lineage markers include any cell lineage markers located inside the cell that can be detected externally. Once incorporated into a cell, the label is reduced or diluted by cell division in that cell. With each cell division, a portion of the marker is transferred to daughter cells. The more often the cell divides, the more diluted the label. In the case of labeled DNA, the proportion of labeled cells transferred to daughter cells is half that of mother cells due to the semi-reserved replication of DNA. After discontinuous administration of cell lineage markers to tissues or individuals, 84752 200401826 cells of tissues or individuals are allowed to divide for a period of time sufficient to produce the first population of unlabeled retention cells, which divides faster than the second population of LRCs and stem cells In order to reduce the concentration of markers of cell lineage present in undivided cells by cell division to reduce the concentration of LRCs and stem cells that are more slowly dividing. In one format, cells of the first population remain undetectable. After label-free retained cells (ie, fast-dividing cell populations) have been diluted by cell division to mark the cell lineage to a concentration below LRCs and / or stem cells, the labeled LRCs and / or stem cells are detected and isolated. These LRCs and stem cells can be identified by antibodies specific for annotation labeling, and separated by fluorescence-activated cell sorting (FACS) or other methods known in the art, because LRCs and stem cells are more effective than non-LRCs. A large number of externally detectable markers are separated based on this feature. In one variation, the administration of the cell lineage annotation marker is interrupted, allowing the cell to divide so that the first population of cells does not contain a detectable marker. In a format, the tissue includes the colon, breast, small intestine, cervix, prostate, skin, bone marrow, liver, heart, bone muscle, thymus, thyroid, thyroid gland, bladder, lung, bile duct, ovary, testicle, brain, lymph Tissue, or other stem cell-containing tissue. After the knife is removed, the LRCs and / or stem cells can be further analyzed and the special biochemical markers on the stem cells can be discarded. Biochemical markers can then be used to isolate stem cells. In addition, LRCs and / or stem cells can be analyzed for DNA damage, mutations, or other chemical changes that may be caused by carcinogen exposure, DNA repair capabilities, oxidative damage, mutation risks, or other genotoxic exposures. In the method of the present invention, the method can be used to identify the chemical agent as being genotoxic to 84752-10-200401826 LRCs and / or stem cells. By administering the chemical agent to tissues or individuals and then detecting DNA modification, including Chemical modification of DNA, DNA cross-linking, DNA crying, base deletion, base insertion, and embedding of isolated LRCs and stem cells. The proliferation rate of LRCs and / or stem cells can also be measured after completing their isolation. In one format, the method of the invention further comprises administering or contacting the tissue or individual with an isotope-labeled DNS synthetic precursor, and incorporating the tissue or individual into the DNA when the cell divides. Isotope-labeled DNS synthesis precursors can be administered to or in contact with tissues or individuals before, during, or after administration to cells. In this method, the isotope-labeled DNS synthesis precursor can be any known DNA synthesis precursor, including (but not limited to) 3H-dT, 2H-glucose, and 2h20. After administering to the tissues or tissues or contacting the precursors for the synthesis of quantitative isotopically labeled dns for a sufficient time to allow the DNA to be labeled in the cells of the tissue or individual, the labeled DNA can be isolated by procedures known in the art. Once the DNA is separated, the isotopic increase of the DNA or its hydrolysis or decomposition products can be measured by mass spectrometers, liquid scintillation counting, gamma counting, nuclear magnetic resonance, and other methods known in the art. Based on this measured increase in isotopes in the isolated DNA or its products, the proliferation rate and rotation of isolated LRCs and / or stem cells can be calculated. In another format, one or more hydrolysates of DNA (such as deoxyribonucleic acid) can be measured. The hydrolysate can also be chemically modified. Isotope increase can be detected by analytical methods, including f-spectrometer, liquid flash 84752 -11- 200401826 isotope increase I count, gamma count, and nuclear magnetic resonance spectroscopy. In particular, the addition can be detected by a mass spectrometer. The tissue can be colon, breast, small intestine, cervix, prostate, skin, bone marrow, liver, heart, skeletal muscle, thymus, thyroid, pancreas, bladder = lung, bile duct, ovary, testes, brain, or lymphatic tissue. The population of cells in a population In addition, the method may further include calculating a population expansion factor. The method of the present invention finds the factors that are used to detect the growth and development of cancer; monitors cancer treatment, monitors the impact of cancer (i.e. carcinogenesis) · monitors the rate of DNA damage fixed to permanent mutations (i.e. mutations, abnormalities Shanghai / adult, carcinogenic effect, evolution rate); monitor T cell response to antigen stimulation (ie vaccine efficacy), monitor and design spermatozoa (ie male fertility ^ pre-adipocyte fat regeneration (ie, natural body fat accumulation)) ; Monitor the maintenance of the total number of epithelial cells (i.e., tissue balance in the body), monitor the expansion of the pancreatic cell group as a marker of the risk of developing diabetes; monitor the expansion of the T-lymphocyte group as an approaching immune agent in progressive lymphopathy such as mV / AIDS Damage markers; monitoring bone expansion of stem cell populations as markers of damaged bone marrow retention; monitoring cell population expansions as markers of impaired tissue retention by replicated consumption; and monitoring the expansion of transplanted bone marrow cells (transplanted cells) as transplant status or Transplant and host disease markers. Kits are also provided to implement the methods disclosed herein [Implementation methods] I. General technology Unless otherwise stated, the implementation of the present invention will make use of molecular biology (including weights of 84752-12-200401826 group technology), microbiology, cell biology, biochemistry, immunology, protein Kinetics and conventional techniques for mass spectrometry are within the scope of those skilled in the art. Such techniques are fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook Et al., 1989) Published by Cold Spring Harbor; Oligonucleotide Synthesis (MJ Gait, 1984); Methods in Molecular Biology, published by Humana; Cell Biology: Lab Notes (Cell Biology: A Laboratory Notebook) (Edited by JE Cellis, 1998) Academic publishing; Animal Cell Culture (Edited by RIFreshney, 1987); Introduction to Cell and Tissue Culture (Introduction

to Cell and Tissue Culture (JP · Mather and P · E · Roberts, 1998) Published by Plenum; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG · Newell, 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Publishing Company); Handbook of Experimental Immunology (DM · Weir and CC Blackwell); mammalian cells Gene Transfer Vectors for Mammalian Cells (edited by JM Miller and M.P. Calos, 1987); Current Protocols in Molecular Biology (edited by FM Ausubel et al., 1987) PCR · The polymerase chain reaction (PCR: The Polymerase Chain Reaction) (Mullis et al., 1994); Current Protocols in Immunology (JEColigan et al., 1991); Molecular biology Short Protocols in Molecular Biology (Wiley 84752 -13-200401826 and Sons, 1999); and eight-year mass isotope isomerism Distribution analysis ·· Mass isotopomer distribution analysis at eight years, theoretical, analytical and experimental consi-derations, authors Hellerstein and Neese (Am J Physiol 276 (Endocrinol Metab · 39) El 146 Yuan El 162, 1999). Furthermore, unless otherwise specified, the use of commercial analysis kits and reagents will generally be used according to the protocol specified by the manufacturer. II. Definitions Unless otherwise specified, all terms used in the techniques, notes, and other scientific terms are used to have a common meaning understood by those skilled in the art related to the present invention. In some cases, terms that have a common understanding are defined herein for clarity and / or ease of reference, and the content of this definition in this article should not necessarily be interpreted to represent a substantial difference from what is commonly understood in the art. The general techniques and procedures described or described in this article are generally well known and generally used by those skilled in the art using conventional methodologies, such as, for example, the analysis of isotope isomer distributions in eight years of mass: theoretical, analytical, and experimental considerations ( Mass isotopomer distribution analysis at eight years: theoretical, analytic and experimental considerations), by Hellerstein & Neese (AmJPhysiol 276 (Endocrinol Metab. 39) E1146- E1 162, 1999). Where appropriate, unless otherwise specified, procedures for using commercially available assay kits and reagents are generally performed in accordance with experimental protocols and / or parameters specified by the manufacturer. Stem cells-Stem cells are slowly dividing progenitor cells that have the ability to self-regenerate and differentiate into mature tissues by forming daughter cells (non-stem cells) 84752 • 14-200401826. Stem cells, also referred to herein as long-term labeled retention cells (LRCs, 10 ng-term label_retaining cells), refer to the slow-proliferating cells of the lineage annotation mark and isotope labeling of the retained cell lineage. Embryonic stem cells are prototype stem cells, which are enough to differentiate to form the entire cell type found in mature animals. Such stem cells are pluripotent because they are capable of differentiating into many cell types. Examples of stem cells include, but are not limited to, bone marrow stem cells, epidermal stem cells, hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, epithelial stem cells, digestive tract stem cells, skin stem cells, neural stem cells, liver progenitor cells, endocrine gland progenitor cells, and Lymphoid-hematopoietic stem cells (which can differentiate into lymphocytes, red blood cells, and members of the bone marrow lineage). Stem cells can be derived from many sources, including (but not limited to) bone marrow, peripheral or mobile blood, umbilical cord blood, fetal liver tissue, other organ tissues, skin, and nerve tissue. As used herein, stem cells include all long-term label-retaining cells (LRCS). ### Baijiaying—Non-stem cells are cells derived from stem cells that have the ability to differentiate into mature body tissues. Non-stem cells divide faster than stem cells can detect. The non-stem cell retentive cell lineage notes that markers can be detected in a shorter time than stem cells (LRCS).应 应 谱 广 注 记 #Note A cell lineage note is a mark that can be incorporated into LRCs and stem cells to allow its identification. Cell lineage annotation markers can be incorporated into the cell's DNA or into other components of the cell that do not transform when there is no cell division. Examples of cell lineages | master markers include functionalized deoxyribonucleosides such as bromide deoxyuran 1? Nucleus substitution (3 〖(111,1) 1'〇111〇 (16〇 \; 7111 ^ (1 丨 116) and Po-15-84752 200401826 IdU (iododeoxyuridine), which is incorporated into the DNA during cell division. The tidal layer plus an increase in body water means that Percentage of total body water that has been labeled when labeled water is administered. 差 房 #Hydroxygenicity means the ability of a chemical to cause damage to DNA (or DNA damage). Examples of DNA damage include (but not (Limited) The chemical modification of DNA, mutation of DNA, base deletion and insertion into DNA, and insertion into DNA are all known in the art. DNA damage can cause many diseases and discomforts, including but not limited to carcinogenesis. Qi馊 means vertebrate, preferably mammals, and more preferably humans. Mammals include (but are not limited to) humans, farm animals, sports animals, pets, primates, mice, and rats. Clan and one group consumption Means that its stem cells retain its population due to activation and proliferation And eliminate specific cell types. An example of population depletion is the elimination of specific T cells in patients with HIV / AIDS. 羞 势 瞀 # A phenotypic marker is an observable biochemistry of cells, tissues, organisms, or individuals Structure, molecule, function, or behavior. Examples of phenotypes include the physical components, macromolecules, cell surface proteins, metabolism, and behavior of cells, tissues, organisms, or individuals. Population Expansion-Population Expansion means by a specific cell lineage Cell division in progress. History of procreation-Proliferation history means the history of expansion of a particular cell lineage population. # 记 的 # —It means water labeled with heavy isotopes of specific hydrogen or oxygen. Label-16- 84752 200401826 Specific examples of water include 2H2O, 3H20, and 18.0. Sub- # means removing a compound from a mixture of compounds. For example, "isolating one or more stem cells" means from a mixture of one or more stem cells and non-stem cells Removal of one or more stem cells. Isolated stem cells may be accompanied by non-stem cells. With these definitions in mind, the present invention is directed to its rapid action The invention relates to a method for isolating stem cells based on their characteristics. Stem cells The present invention relates to the separation of stem cells by a dual labeling method based on the long-lived central quick-moving characteristics, and the measurement of stem cell proliferation rate, population expansion, or history of proliferation, or any dividing cell population. Method of proliferation rate, population expansion, or history of proliferation. As mentioned above, stem cells are slowly dividing progenitor cells that have the ability to self-regenerate and differentiate into mature tissues by forming daughter cells (non-stem cells). Longevity and its ability to regenerate and differentiate, stem cells (especially epithelial stem cells) are involved in many diseases and discomforts including carcinogenesis and many normal physiological maintenance and repair processes, including tissue healing and replenishment. General model of carcinogenesis The current general model of carcinogenesis involves the control of cell cycle and / or the continuous accumulation of mutations in this TΓ related gene in the cell. It is generally believed that at least 5-6 individual mutations are required to evolve into most cancer cells. So this process of carcinogenesis occurs over many years, or even decades, and needs to be continuous or selective in favor of circulating mutations in cells with a fixed intermediate number of key cells. Furthermore, 'because each mitosis (cell division) increases the fixation during replication 84752 -17- 200401826 DNA damage is a permanent genetic mutation and the possibility of generating new errors in DNA or chromosomes. The rate of cell proliferation itself (division) represents the risk of cancer independence Factors, accompanied by DNA damage agents (mutations). Epithelial Carcinogenesis-The Central Role of Stem Cells Epithelial cancers represent the most common cancers in the modern industrial world, including breast, colon, lung, prostate, pancreas, stomach, esophagus, ovary, endometrium, and cervical cancer. On the surface, paradoxically, most epithelial cells exist in tissues for a short period of time and then die and / or fall off from the surface of tissues. So differentiated epithelial cells will not live enough to accumulate the mutations needed to become a cancer cell population. The answer to this contradiction is that the epithelial stem cells that are present in the tissues during the maintenance-ethnic lineage-must be representative of carcinogenic cells. So epithelial stem cells are considered to be the key to major cancers of public health affairs in the modern world. The subject of cell lineage expansion and proliferation is related to the number of cell divisions performed by any particular cell lineage. The history of cell lineage proliferation is important in many diseases and in normal physiological processes. Cell division within a particular lineage, also known as the population expansion of a cell line, especially stem cells, affects the risk of cancer (i.e. carcinogenesis), the rate at which DNA damage becomes permanent mutation (i.e. mutations, teratogenesis, Carcinogenic effect, evolution rate), T cell response to antigenic stimulation (ie epidemic efficacy), spermatogenesis (ie male fertility), fat regeneration from pre-adipocytes (ie natural body fat accumulation), total number of epithelial cells Maintenance (ie, tissue homeostasis). A · Method for isolating stem cells 84752 -18- 200401826 Stem cells can be used by the host cell lineage lineage system; the standard cell lineage line is marked by the standard § own, and the V line is τ 斫, and detected: 'Remembered The cells are then obtained by isolating the retained cells. 1. Applying a cell lineage annotation marker As a first step, inject one or more cell lineages & ρ or tissue. To;? Leopard e π shows 11 kings. I have given it to the individual, and I have enough time and time to record the A㈢-labeled tissue or individual cells (especially stem cells | general cells) that divide during the labeling period. Do you know that the label is a private 5 'deoxyribonucleotide (cm, deoxy_nbonucleotide). The labeled DNA includes any known DNA in the art. Its own DNA. RNA includes any known nucleic acid including deoxythymidine (dT, and .Ca, deoxyadenine, nuclear (dA, deoxyadenosine), deoxycytidine (dc, and Shizhou). Deoxyuridine (dG, deGXyguanGsine), and deoxyuridine (dU, deoxyuridine). The lineage annotation markers can also be toothed deoxyribonucleosides. Deoxyribonucleosides can include any Dental deoxyribonucleosides, including (but not limited to) dT, dA, dC, dG, and dU. A clear example of a notation label for a halogenated cell lineage is an i-deoxyribonucleoside such as brominated deoxyuracil riboside (Brdu, b⑺ deoxyuridine), iodized deoxyuridine core (Idu, i〇d〇de〇xyuridin e), and nucleated deoxycyte 4 p nucleation: y: (BrdC, bromodeoxycytidine). Cell If-based annotation markers can also be radiation-labeled ribonuclei: y :, which can be detected from intact extracellular cells. Radiation The label can be any radioactive isotope known in the art. These include halogen radioisotopes such as Br82-BrdC, Br82-BrdU, 84752 -19- 200401826

Br82-BrdA, lower, and d 82 —annular gas I Γ _BrdG. Other radiation-labeled ribose ribose includes aluminized ribose ribozymes, such as 3H_dc, 3H_dG, 3Y ^, Go_dT, and 3H-dU. The fine marks can also be 気 marks, which can be measured from the complete fine pain. Specific examples include tritium-labeled DNA synthesis precursors such as glucose, and deuterium-labeled ribonucleosides such as 2H_dT, 2 d 2. , And according to the methods commonly used in the art, using physiology and Xu Qing's soil} Wang Han's urine-acceptable solution will be prepared for in vivo cell lineage annotation marks. Depending on the appropriate route of administration. Appropriate routes of administration may include, for example, oral, rectal, transmucosal, transdermal, or intestinal administration; parenteral administration, including intramuscular, subcutaneous, intraspinal injection, and intraspinal, direct intraventricular , Intravenous, intraperitoneal, nasal, or intraocular injection. Cell lineage annotation markers are available immediately, for example, from Sigma Chemical Company of the United States. If one body is introduced, the cell lineage annotation can be administered, for example, at a concentration of 1 mg / ml in drinking water. If other cell lineage annotation markers are used, the toxicity of the application can be easily determined by those skilled in the art. Cellular lineage marker 1 has been administered for a period of time sufficient to incorporate into the organism's DNA. Preferably, the cell lineage annotation markers are administered to a steady state concentration in the tissue or organism. For example, cell lineage annotation markers can be administered to 2, 4, 8 questions or more, as depicted in FIG. Alternatively, the lineage annotation markers can be administered in a local rather than a systematic manner. For example, cell lineage annotation markers can be administered directly into specific -20-84752 200401826 tissues via injection, usually as a long-acting injection or a sustained release formulation that is continuously released at a concentration of 1 mg / ml in water. Similarly, cell lineage annotation markers can be administered until there is a fixed increase in body water in the tissue or individual's DNA. Administration of cell lineage annotation markers can be interrupted prior to isolation of LRCs and / or stem cells. 2. Detect cell lineage annotation markers-Once incorporated into cells, cell lineage annotation markers are reduced or diluted by cells dividing in that cell. With each cell division, some cell lineage i markers are transferred to daughter cells. The faster the cell divides, the faster the cell lineage annotation markers are diluted. After discontinuing the administration of cell lineage annotation marks to a tissue or individual, 'let the tissue or individual cells divide for a sufficient time to produce non-stem cells that divide faster than stem cells to reduce the amount of cell lineage annotation marks to a minimum At slower dividing LRCs and / or stem cell concentrations. The administration is interrupted for a sufficient period of time to form cells of the first group and cells of the second group, wherein the cells of the second group contain more detectable cell lineage markers than the cells of the first group. Cell lineage The administration of annotation markers can be for a limited amount of time. For example, administration of cell lineage annotation markers can be interrupted for 1 week, 2 weeks, 4 weeks, or 8 weeks. After the non-stem cells have been diluted by cell division to mark out the cell lineage markers to a concentration lower than the LRCs and / or stem cells, the LRCs and / or stem cells can be identified. Cell lineage markers can be detected using antibodies that specifically identify the lineage markers. An "antibody" (hereinafter used in plural) is an immunoglobulin molecule that can specifically bind to a target such as a cell lineage marker (by 84752 -21-200401826 by at least one antigen located in a variable region of the immunoglobulin molecule) Identification area). As used herein, this term includes not only whole antibodies, but also fragments thereof (such as Fab, Fab ', F (ab') 2, Fv), single chain (ScFv), mutants thereof, and fusions comprising an antibody portion Modified structures of proteins, artificial antibodies, and any other immunoglobulins that contain the required specificity for antigen recognition. The antibody may be single or multiple. For example, BdU, IdU, or 2H or 13C labeled DNA, or other cell-labeled monoclonal antibodies can be used to identify cell lineage labeling. Anti-BrdU and anti-IdU monoclonal antibodies can be purchased from phar-mingen and Resaerch Diagnostics. BrdU or IdU-labeled stem cells and non-stem cells and BrdU or IdU incorporated with a specific anti-Brdu or anti-Idu monoclonal antibody, respectively, can be followed by well-known procedures in the art. 3. Isolate stem cells and then isolate cells containing cell lineage annotation markers. Labeled cells can be separated by fluorescence-activated cell sorting (FACS) and other methods known in the art. FACS is a technology based on the amount of fluorescent light emitted at a specific wavelength, used to separate and sort cells marked with a fluorescent marker (such as a cell lineage marker). The FACS method is described in, for example, flow cytometry: a practical method (FiOw

Cytometry: A Practical_pproach. M.G. Ormerod, 2nd edition, 1994). In another embodiment, irradiated isotope-labeled cells such as liquid scintillation counts or gamma counts can be observed outside the cells. Mass-based separation techniques, such as centrifugation, can also be used for isotopically labeled cells. 84752 -22- 200401826 can isolate LRCs and / or stem cells from tissues including colon, breast, small intestine, cervix, prostate, skin, bone marrow, liver, heart, sclerotomy muscle, thymus, thyroid, pancreas, bladder, lung, Bile ducts, ovaries, testes, brain, lymphoid tissue, or other tissues that may contain stem cell populations. From, his method can be known; For example, green fluorescent protein (GFP, Greenfluorescent protein) is transiently retained in stem cells, but is diluted with each cell division. GFp is retained in stem cells but disappears in rapidly dividing non-LRCs. Transiently expressed proteins such as GFp can be used as cell lineage annotation markers, or can be used for the purposes of the invention disclosed herein. B. Measuring the Proliferation Rate of Stem Cells In addition to the isolation of LRCs and / or stem cells, the proliferation rate of coffee or stem cells can be determined. The proliferation rate of LRCs and stem cells affects the risk of cancer ^ carcinogenesis), the rate at which fixed DNA damage becomes a permanent mutation (ie mutation occurrence, teratogenesis, carcinogenesis, evolution rate), the response of τ cells to antigenic stimulation (ie vaccine efficacy ), Spermatogenesis ability (ie male fertility), fat regeneration from pre-adipocytes (ie natural accumulation of body fat), preservation of total epithelial cells (ie, tissue internal balance), and other medical conditions and diseases. The involvement of cancer epithelial cells that cause solid tumors in the human body to organs such as the lungs, breasts, skin, mouth, and colon is known as cancer. Cancers involving human epithelial cells can form solid tumors from the breast, lung, stomach, liver, uterus, colon, skin, mouth, and cervix. Glands J from secretory tissue and squamous carcinoma from protective lining are two basic types of cancer. The above two cells are basic cancers that rapidly proliferate regardless of cell boundaries. Because many cancers originate from epithelial cells, there is great interest in being able to measure the growth rate of epithelial LRCs & or stem cells = 84752 -23- 200401826. The rate of proliferation of LRCs and / or stem cells can be measured by the dual labeling method. Both cell lineage annotation labels and different isotopically labeled DNA synthesis precursors are administered to the cells. Isolate LRCs and / or stem cells by detecting long-term cell lineage annotation labeling resident cells, and increase the isotopes of DNA to measure. The proliferation rate, population expansion, and proliferation history can then be determined or monitored. 1. Administration of a cell lineage annotation marker As a first marker, as described above, one or more cell lineage annotation markers are administered to a tissue or an individual. Administration can be interrupted. 2. Administer an isotope-labeled DNA synthesis precursor as the first label 1 ′ before synthesizing one or more isotopically-labeled dNA

Other amino acids, and J sulfonate. $ 102, NH3, urea, 02, ketone bodies and fatty acids, glycine, 02, glucose, lactic acid, succinate or 84752 -24- 200401826 isotope-labeled DNA synthesis precursors can also include one or more Nucleoside residues. The DNA synthesis precursor may also be one or more components of a nucleoside residue. For example, glycine, aspartic acid, glutamine, and tetrahydrofolate can be used as precursor molecules for the p-epicyclic ring. For example, aminomethyl phosphoric acid and aspartic acid can be used as precursor molecules of the pyrimidine ring. Adenine, adenine nucleoside, guanine, guanine nucleus can be administered as a precursor of DNA synthesis. All isotopically-labeled DNA synthesis precursors are commercially available, for example, from Cambridge Isotope Labs (Andover, MA). The DNA synthesis precursor can be water. DNA synthesis from 2H20 can be measured using polyribonucleosides, polynucleosides, and C-H bonds of ribonucleosides or nucleosides. The C-H bond undergoes deoxyribonucleoside exchange from H20 into the cell. The presence of the 2H tag in the C-H bond of polyribonucleosides, polynucleosides, and ribonucleosides or nucleosides after 2H20 administration indicates that DNA was synthesized during this time. The degree of labeling present can be determined experimentally, or it can be assumed based on the number of labeling sites in the DNA. For example, Figure 2 depicts the incorporation of deuterium into DNA's deoxyribose from water. Hydrogen atoms from body water can be incorporated into free nucleosides or polyribonucleosides. 2H or 3H from labeled water can enter these molecules via an intermediate metabolic reaction.

Those skilled in the art will know that hydrogen atoms from body water can be incorporated into other polyribonucleosides, ribonucleosides, or nucleosides through many biochemical pathways. For example, glycine, aspartic acid, glutamine, and tetramethylfolate are known precursors of the purine ring. For example, carbamate phosphate and aspartic acid are known precursors of the pyrimidine ring. Ribose and ribose phosphate, and their synthetic pathways are known precursors synthesized by DNA 84752 -25-200401826. Through biochemical reactions catalyzed by enzymes, he, "the polynucleus is called ribose ribobo, = two = ziyan 'group-guanine, and other oxidative test bases or nucleus oxygen atoms from 1802 are incorporated into ribonucleosides. It is also known that its precursors for DNA synthesis can also include isotope-labeled amine di :. Isotopically-labeled amino acids include (but are not limited to) I3C-lysine, naphthalene, other, labeled Or 13 ° -labeled amines and deuterated amino acids. 2 In particular, kappalitin-labeled DNA synthesis precursors can be glucose or H20, such as U.S. Patent No. 5,91MQ3 & 6, gig, 846 ^^. The detection of the isotope-labeled DNA synthesis precursor can be discontinued before the detection of the cell line annotation label or the measurement of the isotope increase of the DNA. 3 · Detection of the cell lineage annotation label is as described above, and then one or Isolate one or more cells labeled with various stem cells. 4. Measure the increase in isotopes of DNA. Obtain DNA from the cell populations studied and use standard analysis techniques to analyze DNA molecules that can be incorporated into the labeled DNA precursors as described above. Chemistry Department The amount of isotopes in the cell determines the amount of stable isotope A incorporated into the DNA of the cell. Examples of techniques include, for example, mass spectrometers, and nuclear magnetic resonance, and liquid scintillation counting. Sample preparation methods will be used to detect the presence of isotope labels It depends on the specific analysis technique and will be apparent to those skilled in the art of this -26- 84752 200401826. The DNA of cells containing cell lineage annotation markers can be partially purified or isolated from the cells. It can be done by any method known in the art Obtained from cells, as described in Molecular Colonies: A Laboratory Manual L ^ axaloxyl Shuai-), (Sambrook et al., Cold Spring Harbor, 1989). The consistent method of DNA isolation will depend on the specific cell type, and It will be apparent to those skilled in the art. DNA can be hydrolyzed to deoxyribonucleosides using standard hydrolysis methods, such as those well known in the art. For example, DNA enzymes can be hydrolyzed, such as, for example, with nucleic acid enzymes or phosphate enzymes, or with acids and bases. Or other chemical hydrolysis methods other than enzymatic hydrolysis. As mentioned above, after partial purification or single isolation by any known separation method, the hydrolysis can be measured as appropriate. Products. The incorporation of DN A-or multiple isotopic labels can be detected by many methods, such as mass spectrometers (including but not limited to gas chromatograph_mass spectrometer (gc_ms, office chrmatography_mass spectr〇metry), isotope ratio mass spectrometry Analyzer, GC-combustion isotope ratio · MS, splitting_isotope ratio capture, liquid chromatography_MS, electrospray free_MS, matrix-assisted laser desorption free time of flight-MS, Fourier transform ion acceleration Resonance ru, cycloid_ms), nuclear / resonance (NMR, nuclear magnetle res_nee), and liquid flashing count. a • The mass spectrometer detects the isotope by the mass spectrometer. National Patent No. 5,910,403 and in the preferred embodiment of the present invention, the presence of the label, as described in, for example, US 6,010,846 〇84752 -27- 200401826. The constituents become fast-moving gas phase ions and are separated based on their mass-to-charge ratio. The distribution of isotopes or isotopes of ions can therefore be used to measure the increase in isotopes in cellular DNA. Generally speaking, mass spectrometers include ionization tools and mass analyzers. Many different types of mass analyzers are known in the art. These include (but are not limited to) sector magnetic field analyzers, electrostatic analyzers, quadrupole columns, ion traps, time-of-flight mass analyzers, and Fourier transform analyzers. In addition, two or more mass analyzers can be connected (MS / MS) to separate precursor ions, followed by gas phase fragment ions. Mass spectrometers can also include many different ionization methods. These include, but are not limited to, gas-phase ion sources such as electron impact, chemical ionization, and field ionization, and desorption sources such as field desorption, rapid atomic impact, matrix-assisted laser desorption / dissociation, and Surface-enhanced laser desorption / release. In addition, the mass spectrometer can be connected to separation tools such as gas chromatography (GC) and high performance liquid chromatography (HPLC). In a gas chromatograph mass spectrometer (GC / MS), connect the capillary column from the gas chromatograph directly to the mass spectrometer, using a jet separator as appropriate. In this application, a gas chromatography (GC) column separates sample components from a sample gas mixture and ionizes the separated components and chemically analyzes them in a mass spectrometer. When GC / MS is used to measure the mass isotope isomer content of organic molecules, the hydrogen-labeled isotope from the labeled water is enlarged by 3 to 7 times, depending on the number of hydrogen atoms incorporated into the organic molecules from the labeled water. It depends. In another embodiment, the isotope-labeled -28- 84752 200401826 DNA may be partially purified or isolated as appropriate prior to mass spectrometric analysis. Furthermore, isotopically labeled hydrolysis or decomposition products can be purified. / In another embodiment, the isotopic increase of the isotope-labeled DNA after hydrolysis is measured by a gas chromatograph mass spectrometer. Using standard techniques (such as synthesizing trimethylsilyl, methyl, acetamyl, etc., directly > Wang into the liquid chromatography; and direct detection of sample introduction) can be prepared for mass spectrometry Analyze and determine the amount of label incorporated into deoxyribonucleosides. It was analyzed that it may contain stable isotopes introduced from the endogenous marker pathway. For example, after administration of glucose at 6,62, using a gas chromatograph and a micrometer under free electron impingement and a selected ion recording mode, the deoxyadenosine of the + positive deoxyribose ring can be changed. Mass spectrometric analysis of the 467-469 nuclear fragment or the m / z 5 57-559 fragment of deoxyguanosine. b · Liquid scintillation Li3 can observe the radioisotope with a liquid scintillation meter. Radioisotopes such as radon emit radiation and are detected by a liquid scintillation detector. The detector turns into a packet message and zooms in. Thus, the number of radioactive isotopes in a cell or DNA can be measured. In one or more embodiments, the isotope-labeled DNA can be irradiated, and the added value of voxel can be directly measured by liquid scintillation. In another specific embodiment, the radiation isotope may be 3h. In another embodiment, the isotope-labeled DNA or its components can be purified or isolated as appropriate, and then measured by liquid scintillation counting. 5. Calculate the proliferation rate and population expansion 84752 -29- 200401826 In each of the above specific embodiments, by using precursors to increase the mass or asymptotic isotopes with the isotope to increase the countable relationship, benefit or the rate of stem cell biosynthesis or decomposition "t In addition, ^, 'I's LRCs and / or have reduced the curvature rate by using exponential or other decelerating models. N # biosynthesis or decomposition rate of rapid proliferation "and" rotation rate "can be isomerized by mass isotopes 'Physical analysis' is calculated by hand or by calculation. Briefly, a portion of the newly synthesized DNA is replaced (%) at each time during the administration of an isotope-labeled DNA synthesis precursor from an excessive amount. θ Partial replacement of newly divided cells at various time points during the administration of-or multiple isotopically labeled DNA synthesis precursors (f) is determined by the following formula: M + 0 + M Guangpin) content f (of new cells Partial ^ Ssxio. Where M + 0 is the parent mass isotope isomer of the derived dR; M + 1 is the mass plus-isotope isomer of the derived dR; EMI is an excessive content of the mass isotope isomer. For example, It is known in rodents that colon cells are completely replaced by new cells within 6-8 days. Tissues or individuals can be maintained for one or more isotopically labeled DNA synthesis precursors for a period of time, such as 2 to 3 weeks, to ensure complete replacement and use of their %% Replaced cells increase as a comparison (denominator) or asymptotic value. The replacement rate constant (k) can be calculated from the formula: k = -ln (1 -f) / t where k = rate constant (cT1) and t = labeling period ( Days) -30- 84752 200401826 ti / 2 (half-life) = 0.0693 / k An example of mass isotope isomer analysis is Mass Isotope Distribution Analysis (MIDA) or combined analysis. This Algorithm described in Xu Those skilled in the art known to the different source of clear terms, MIDA was calculated at US Patent No. 5,336,686 of the theme. This method is further defined by Hellerstein and Neese (1999), and

Chinkes et al. (1996), Kelleher and Masterson (1992), and Macallan et al. (1998), and Neese (2001). In addition to the references cited above, the calculation software that implements this method is commercially available from Professor Marc Hellerstein (University of California, Berkeley). It is also possible to use the average population expansion factor among different populations. The average population expansion (C.E.) factor system is defined by the following formula: u speed The population expansion factor is used to compare the proliferation rate or rotation rate between different cell populations. The rate constant k is equal to CE / t, where t is the marking period. Therefore, k is the rate constant, the rotation rate, or the proliferation rate measured at each time point. + The population expansion m sub is used to calculate the proliferation history or fine line. Population Expansion The number of cell populations in Adachi has expanded exponentially over a period of time. Therefore, the factory can use the ethnic expansion factor to compare the "proliferation rate or rotation speed T" between the same ethnic groups. For example, some cells can rotate at a rate of 20% per day, and other days; two at a rate of G.7% per day. The population expansion of different cell populations can have different population expansion factors, which can be compared. C. Using precursor molecules to synthesize precursor molecules from mosquito stem cells. Using mono-isotopic labeling of DNA to synthesize precursors. Using 84752-31-200401826 labeling methods, the presence of LRCs & or stem cells can be determined. In this method, an isotope-labeled synthetic precursor is applied to a tissue or individual, as described above. As appropriate, maintain a steady increase in the amount of DNA synthesis precursors. § When fe / f DNA synthesizes precursors and measures the increase of isotopes of DNA in tissues or individuals in different cells, cells that retain an increased amount of isotopes are considered as LRCs and / or stem cells. By monitoring the same tissue over time, the stem cell proliferation rate and population expansion can be measured, as described above. This one labeling method is different from the above dual labeling method and cannot isolate LRCs and / or stem cells. It can only determine the presence, absence, or content of LRCs and / or stem cells in the tissue. Use of stem cell isolation and proliferation rate measurement The method of the present invention finds use in detecting and quantifying cancer growth and development; in monitoring the treatment of cancer, monitoring the risk of affecting cancer (ie, canceration), monitoring the damage of fixed DNA to become permanent mutations Rate (ie, mutation, ovarian-induced fetality, canceration, evolution rate), monitoring T-cell response to antigenic stimuli (ie, vaccine efficacy), monitoring and designing spermatozoa treatments (ie, male fertility); preadipocytes Fatty neonatal (ie, natural accumulation of body fat); monitoring the maintenance of the total number of epithelial cells (ie, tissue homeostasis); monitoring the expansion of the pancreatic β-cell population as a marker of the risk of developing diabetes; monitoring the expansion of the T lymphocyte population as a gradual progress Lymphatic diseases such as HIV / AIDS approaching immunological damage markers; monitoring bone and stem cell population expansion as markers of damaged bone marrow retention; monitoring cell family monarch expansion as markers of impaired replication consumption of damaged tissue retention; and monitoring : The population expansion of the transplanted bone marrow cells (transplanted cells) as the transplantation state or transplantation and host disease (GVHD, graft vs. host disease). -32- 84752 200401826 Once the LRCs and / or stem cells are isolated, they can be given properties using procedures well known in the art. For example, LRCs and / or stem cells can be analyzed with a program exemplified by DNA technology and detection (Technologies for Detection of DNA Damage and Mutations. GP Pfeifer, Plenum Press, 1996) to determine whether they DNA is modified in any way. Cell modification may result from carcinogen exposure, DNA repair capacity, oxidative damage, mutation risk, or other possible genotoxic exposures, and its effects can be measured. The methods described herein can be used to identify diseases or disorders. For example, this method can be used to identify and quantify canceration. Stem cells are important in colon cancer because they can be the earliest ancestors of cancer cells. For example, see Bj erknes, M (1996) Expansion of mutant stem cell populations in the human colon. colon, J Theor Biol 1 78, 38 1-385). Due to their characteristics such as slow proliferation rate, longer life in tissues, high proliferative potential, and multiple nature, colonic stem cells are considered to be the main cell type that has the risk of accumulating mutations that cause cancer. See, for example, Bach, S.P., Renehan, A.G., and Potten, C.S. (2000), Stem cells: the intestinal stem cell as a paradigm. Carcinogenesis 27, 469-476. In contrast, other normally differentiated cells are short-lived. When a mutated stem cell undergoes population expansion, the risk of subsequent daughter cell mutation increases and makes the cell easier to start. The methods described herein can also be used to anchor other diseases or disorders. For example, this method can be used to identify the risk of diabetes, or the efficacy of treatment, by using pancreatic β-cells. This method can also be used to identify immune impairments, for example by measuring T-cell progenitors to identify progressive lymphopathy, such as AIDS. The method can also be used to identify transplantation and host disease (GVHD, graft vs. host disease) by measuring the onset of transplant rejection by measuring τ cell proliferation. LRCs and / or stem cells with altered proliferation rates can be used to determine DNA modification. Examples of DNA modification include, but are not limited to, chemical modification of DNA, DNA cross-linking, DNA mutation, base deletion and insertion into DNA, and embedding in DNA, all of which are known in the art. Alternatively, this method can be used to assess the genotoxicity of putative genotoxic agents. By isolating LRCs and / or stem cells, or measuring the proliferation rate of LRCs and / or stem cells, and correlating LRCs and / or stem cells or proliferation rates with DNA modification by methods known in the art, putative genes can be Evaluation of the effects of toxic agents. The methods described herein can also be used to identify stem cells and can be used to identify stem cell phenotypic markers. Phenotypic markers include any observable biochemical structure, molecule, function, or behavior of LRCs and / or stem cells. Once the LRCs and / or stem cells are isolated, they can be studied to determine if there are any other phenotypic markers. Alternatively, LRCs and / or stem cell phenotypic markers with altered proliferation rates can be studied to identify phenotypic markers. The methods described herein can also be used to identify therapeutic compounds. For example, a putative therapeutic compound can be administered to a tissue or individual, and then or together to a stem cell lineage-labeled and / or isotope-labeled DNA synthesis precursor. The effect of the putative therapeutic compound can then be monitored. The putative therapeutic compound may be a chemical or a drug. Putative therapeutic compounds may also be dietary factors, including soy-derived products such as genistein and lunasin, rapeseed-derived products, and antioxidants such as vitamin C, vitamin E, and vitamin A. LRCs and / or stem cells from treated tissues or individuals can be compared to -34- 84752 200401826 to identify the effects of putative therapeutic compounds on untreated tissues or individuals. This method can also be used to identify compounds that stimulate stem cell proliferation. For example, after administration of a putative lymphocyte synchronizing stimulus, tau cells in a tissue or individual can be monitored to observe the effect of the synchronizing stimulus. Alternatively, you can measure: Copy consumption. The invention will be better understood by referring to the following non-limiting examples. Example development-A dual-labeling method using brominated deoxyurinary nucleus biting nucleus bitumen (bromideoxyluridine) (thymic analogs) as a marker for labeling retained cells' (LRCs) and glutamate, Using gas chromatography_mass spectrometer (gc_ms, material s

Chromatography-mass spectrometer) determines its proliferation rate. A schematic illustration of the labeling experiment is shown in FIG. V. Animals Fisher 344 rats (male, four weeks old at the beginning of the study, Simonsen, San José, CA) were used. All procedures were approved by the University of California, Berkeley Experimental Line Support Office. HOS flats consist of 3 large white Tibetans. Diet: Purina rat food, unlimited supply. Maintain twelve hours light-dark cycle. At each measurement time point, the rats were euthanized with a dioxide suffocation method. There was no significant difference in the weight of the animals given the tear water compared with = (no BrdU in the drinking water). Animals were divided into three groups and received prices of £ 11 for 2, 4, and 8 weeks; in drinking water. Then rats were administered with 8% 2 ^ and demarked at 0, 2, 4, and 8 weeks 3, and sacrificed after the 0 mark period (4-9 rats each at the demarking time point) (Figure 1). 84752 -35- 200401826

BrdU administration For BrdU labeling, rats received βΗυ at 1 mg / mi in drinking water. Glucose (1 g / 100 ml) was added to the water to mask the taste of BrdU. Add Brdu to the water and shake the bottle. Next, add glucose and shake the bottle vigorously to completely dissolve BrdU. It takes i_2 minutes to completely dissolve the Brdu. It is important to prepare fresh BrdU drinking water daily during feeding. 2h2o administration At the beginning of the he labeling period, rats were injected intraperitoneally with 99.9% 2%. The injected limb sum is 4% of the estimated body water volume. It was then maintained at 8% 2H2O and drinking water was supplied unrestricted for 8 weeks (19). Increased body water Figure 2 shows a diagram of deuterium ribose (dR) in which deuterium is incorporated by water. Body moisture increase is measured by the gas chromatography / mass spectrometer (gc / ms, gas spectrometer) technique described above. Isolation of epithelial cells (CEC, Colon epithelial ee [l) Isolate CEC from the bottom of the glandular fossa, this is where LRCs are assumed to exist, and the nucleus is fixed and stained with a single anti-Brdu FITC antibody for flow cytometry. The colon was immediately removed at the time of dissection. The feces were removed with 0.05 M NaCl with 0-〇001 M dithiothreitol (DTT, dithiothreitol) and the colon was placed in cold 1.6% Joklik's modified medium (jmmEM, Joklik's modified medium, Gibco Invitrogen, Grand Island, NY) 120 minutes. Later, non-enzymatic and mechanical separation methods described elsewhere were used to separate CECs from different proliferative fractions in the glandular fossa. CECs from different glandular fossa parts (bottom proliferative part and 84752 -36- 200401826 top thermogenic part) were collected by culturing the sacs filled with chelating agent-containing pB $ buffer. The tissues were shaken in a 37 ° C water bath for 20 minutes, then 15 minutes and 30 minutes to collect CECs from the top, middle and bottom sections, respectively. Discard the middle points (transitions). The collected CECs were then separated from intraepithelial lymphocytes and other contaminants by gradient centrifugation (covered with 45% paco on 75% paco) using discontinuous Paco [Percoll, Amersham Biosciences, Uppsala, Sweden]. CECs from the proliferative fraction (bottom of the glandular fossa) were collected and used for FACS and GC / MS analysis because LRCs are known to be located in this area. CECs from mature (top) fractions of rats given 2H2O for 2-3 weeks were used as complete replacement CECs to represent the largest 2H marker in DNA. A portion of the distal and proximal colons was used for immunohistochemistry. Flow Cytometry Analysis of BrdU-labeled Cells The staining procedure was as described elsewhere, and CECs were modified. CECs were isolated from the colon (as described above) and filtered through Falcon tube 2235. The cells were then fixed in 95% ethanol on ice while vortexing, centrifuged at 800 g for 3 minutes and washed once in cold pBs (without Ca ++ and Mg ++). Because CEC showed a tendency to aggregate, the nuclei were removed from the cells for separation by flow cytometry. Two million cells were washed with ethanol in 150 μΐ PBS, incubated in a 1% NP 40 (Sigma) solution for 30 minutes in a refrigerator, centrifuged at 10,000 g, and collected in pBS. Cleaning. Two million CEC nuclei were added to each well of a 96-well plate (round bottom), and 150 μΐ of 1% polyformaldehyde with 0.01% Tween 20 was added to PBS for further fixation and permeabilization. The cells were kept at room temperature for 30 minutes and then at 4. (: 15 minutes, then centrifuge at 800 g for 3 minutes and wash once in cold PBS. Next, add 150 μΐ DNase I (50 k units / ml) at room temperature for 15 minutes at -37- 84752 200401826 Denatured nuclear DNA. After washing the cells with PBS once, 50 μΐ of FITC (fluoresecin isothiocyanate) -linked anti-BrdU monoclonal antibody (Becton Dickinson, Palo Alto, CA) and 0.1 % Tween 20-20 μΐ PBS was added. For the negative control group, equal concentrations of mouse IgGl FITC isotype control antibody (Becton Dickinson, catalog number 3 49041) were used to correct background fluorescence. Cells were stained at 4 ° C overnight, and the next day Wash twice with cold PBS, and then filter with Falcon tube 2235 before analysis. Cells were loaded with a 15 mW argon ion-cooled laser, a He-Ne air-cooled laser, and a water-cooled 5 W argon ion laser. Screening by Beckman-Coultei * EPICS Elite Cell Screener. Cell Proliferation Analysis A) Isolation of DNA from deoxyadenosine (dA) and derivatization of deoxyribose (dR) FACS collects LRCs in free rats CECs and proliferation of white spoon to which DNA is isolated using the Qiagen reagent set (Qiagen, Valencia, CA). The DNA was hydrolyzed to isolate dA as previously described. The isolated dA is excised from the base portion to obtain dR. The dR pentacarbonate-tetraacetate (PTA) was prepared for GC / MS analysis. GC / MS analysis was performed by methane CI using a 30-meter DB-225 column (0-25 mm inner diameter, 0.25 μm film thickness , J & W Scientific, Folsom, CA), using selected ions to detect m / z 245 and 246. B) Calculate the% of newly synthesized cells by comparing fully rotated CECs. The partial replacement (%) of the newly synthesized DNA at each time point during the 2H2O administration period is determined by the following formula: M + o is the parent mass isotope isomer of the derived dR 84752 -38- 200401826; 1 ^ +1 is the mass of the derived dR plus isotope isomers; Ειι is the ratio of M! Molecular pair (M + Q + M + 1) content to the natural content correction of the mass mass of isotopes of Mi, ie M + 1 mass Excessive isotope content. Partial replacement (f) of newly synthesized DNA at various time points during the 2H2O administration period is determined by the following formula: M + 1 (sample) content M + 1 (unlabeled standard) content 1 M + ( ) + M + 1 (sample) content M + () + M + 1 (unlabeled standard) content (part of new cells) X 100 five from 1 (colon cell sample) five (completely replaced colon cells) Where M + ο is the parent mass isotope isomer of the derived dR; “+1 is the mass of the derived dR plus the isotope isomer; EM1, M + 1 is an excess of the mass isotope isomer. It is known that colon cells are completely replaced by new cells within 6-8 days. As mentioned above, the replacement rate constant (k) can be calculated: k = -ln (lf) / t where the rate constant (cT1) and t = labeling period (days) ti / 2 (half-life) = 0.693 / k so rats are maintained at 2H2 02-3 weeks to ensure complete replacement and use their increase in CEC as a comparison (denominator) or asymptotic value (18-20). The increase in body water 2H2〇 during 2H2〇 administration is shown in Table 1. Table 1 Increase in body water 2H20 during 2H20 administration Week 2H20 increase 1 4.29 3 3.93 4 4.30 -39- 84752 2004 01826 Then calculated the proliferation rate of LRC (estimated stem cells) in the colon: f (% new DNA) = 0.33%-0.99% / day ti / 2 = 77_210 days BrdU immunohistochemistry Cut the distal colon (1 cm) and cut Fix in 10% PBS buffered formalin overnight. Fix the fixed tissue in paraffin and cut into 5 micron pieces to fix on glass slides (Histotec laboratory, Hayward, CA). Heat the slides to 56-58 ° C 30 minutes and remove paraffin with Hemo-D remover (Fisher Scientific PA). Tissue was rehydrated with loo% to 70% ethanol. Slides were cultured in 2 N HC1 at 40 ° C. Endogenous peroxidase activity Stop with 3% H2O2 for 15 minutes. Use horse serum blocking solution (Vectastain ABC Elite kit Vector, Burlingame, CA) to block non-specific binding. Then slide the slides with anti-BrdU monoclonal antibody (1:50 Diluted in PBS) (Becton-Dickinson, San Jose, CA) cultured overnight in a humidified room at 4 ° C, and then biotinylated secondary antibodies (Vectastain ABC kit, Vector). Streptavidin was used (Vectastain ABC kit, Vector) to detect and use color 3 3 - a group benzidine (DAB, diaminobenzidine) tetrahydrochloride (Sigma) colorimetric 7-15 minutes. Cells were stained with hematoxylin solution (Sigma) and the slides were stored in permount (Fisher Scienti: fic). A Zeiss (2; eiss) microscope with a video camera and a screen was used to determine the labeling index [(Number of BrdU cells / total number of cells counted)]. For the colon, from each sample to at least 10-15 well-defined glandular fossa (1000-1500 total cells). All cell counts were performed independently by two observers of unknown study design. • 40- 84752 200401826 Agophilic Staining Digestive gland endocrine cells are specifically stained with Agophilic staining. The tissue is fixed in a manner similar to BrdU immunohistochemistry. The fixed hearts were stained with a Fontana-Mass () n anodophilic response method. : Glandular endocrine cells show 絜 彳 on a pink background of cytoplasm. During the period, the body weight of the rats administered to Brdu (oral) was increased normally compared with the control group rats (not shown). The amount of Brdu water consumed per day is 48 ± 12 ru / rats. -41-200401826 and 8 weeks later, 89, 65, and 77% BrdU + cells were identified (analyzed by FACS) (77 ± 12%, mean ± SD).

BrdU-de-labeled (washed-out) CEC distribution chart Figure 58 shows the distribution of 0 ^ (^) distribution from 2 ^ 4 and 8-week 3 ^ 11 unlabeled rats. Using strict criteria, only BrdU peaks were selected to be bright BrdU-collected cells were collected at the top 20-40% of the end. Figure 5B shows that BrdU and BrdU-free cells were reanalyzed by FACS after isolation, and there was no duplication between BrdU- and BrdU-free cells screened by FACS. During each delabeling period Afterwards, the percentage of BrdU cells was measured by flow cytometry. To determine the ratio of LRC in proliferating cell separation, the percentage of BrdU cells was measured by FACS during delabeling (Figure 6). On average, at 0, 2, 4 and After 8 weeks of de-labeling, 77, 12, 7.0, and 3.8% of LRCs (4-9 animals / group) were observed (Figure 6). This proportion of LRCs was similar to that reported by other researchers (6, 8, 17). Utilizing the proliferation rate of LRCs in deuterated water fences, the LRCs were collected by FACS for GC / MS analysis. DNA 2H from LRCs was measured by GC / MS and the kinetics were calculated as previously described (20). Table 2 shows LRC The increase in 2H is reflected in the division of LRC during the 2H20 marker after interruption of BrdU intake. In the total population of LRCs On average, 0.3 3 ~ 0.9% division or ~ 0.8-2.5 million new LRCs are produced every day, assuming that the total number of cells in the colon is ~ 250 million. The average half-life of LRC is calculated to be 77-210 days. 84752- 42- 200401826 Table 2 · Kinetics of LRCs grdU demarked soil ^ 2〇 Period of application ^ __0,90 ti / 2 (days) 77

The BrdU delabeling and simultaneous IQ labeling were performed as described in the text (n = 22). One hundred color of water-based LRCs We also studied rats that were born after being labeled in the womb (the mother administered 1 to 0 throughout the pregnancy). Three young rats were administered 4% Goo for 207 weeks after birth, and then sacrificed (fully deuterated rats). The other 4 rats were stopped and ingested at 7 weeks of age and demarked for 2 weeks (until 1h20 was washed out of their body water), and then sacrificed. Compare the 1h increase between fully deuterated and delabeled CECs. For fully labeled and unlabeled animals at 2 weeks, the EM + ι component ll is 10.53 ± 0.2% and 1.04 ± 0.18%. The EM + 1 value of the unlabeled cells was Yuanquan; 9.9% of the EM + 1 value of the 11 CECs was equivalent to the percentage of LRCs in the unlabeled white blood cells (Figure 7). It is similar to the percentage of BrdU cells that were unlabeled for 2 weeks as measured by FACS. Yuwei liU cancer-free histochemical chemistry of LRC in the fossa gland fossa_ We used BrdU to mark the gland fossa by immunohistochemistry at 4 weeks to determine the location of the LRC (Figure 8B). In Figure 8A, most of the CECs in the fully BrdU labeled gland were marked brown after 2 weeks of BrdU treatment. In the figure, we can see that a cell near the base of the gland fossa retains BrdU. The argophilic staining in the colonic fossa of the colon is because we believe that the number of digestive gland endocrine cells (which is less than total epithelial cells 84752 -43-1 week _4 weeks 8 weeks 200401826) (&1; 1%, 28) Colon cells have a longer lifespan (~ 35-100 days). We depend on the presence of digestive gland endocrine cells in our samples (Figure 9). Negligible amounts of digestive gland endocrine cells are present. Furthermore, it is not found in our study where LRCs are known to exist and are identified (most of them are located at the base of the glandular fossa). Therefore, we can effectively delete this other type of longevity cells that obviously contaminate our LRCs isolated from the colon. The procedure to isolate LRCs from the colon of rats is labor-intensive because LRCs are scarce (3.8-12%) and we collect 20-40% of the fraction above the BrdU peak. Furthermore, CECs are easy to aggregate, so we must process the nuclei on FACS, which is slower than blood cells; the data processing rate is 400_500 times / second vs. 5000 times / second). In order to collect 30,000 to 500,000 nuclei (the minimum number required for each gc / ms analysis), it took 4-12 hours. Improvements in GC / MS technology or instrumentation can reduce the number of cells needed to further study mass spectrometer signals (Hellerstein, MK, Awada, M and Neese, RA, unpublished observations). The simpler and more stable increase of 2H in body water is worth emphasizing. A steady increase in body moisture was maintained during the study period (4.2 ± 0.2% of total body moisture, Figure 3). The results of the FACS profile confirmed that Brdu was effectively delivered to colon cells from drinking water (Fig. 4A'B). After 2 weeks of BrdU administration, approximately 90% of the CECs from the proliferative fraction had BrdU. However, BrdU-reduced cells decreased to 65-77 ° / after 4 to 8 weeks of Brdu administration. . This decrease may be due to the slandering of a liver enzyme that denaminates BrdU as part of animal regulation of long-term administration of Brdu (32). The slander of Brdu-decomposing enzymes makes it difficult for Brdu to obtain and incorporate into cells. The drinking water method (Kouyueliang) is a better method for long-term administration of BrdUr than intraperitoneal injection because 84752 -44- 200401826 is less toxic in animals, but it becomes obviously less effective as a marker over a long period of time. Repeated intraperitoneal injection of BrdU caused weight loss in rats (data not shown) 'although this is a more effective way to deliver BrdU to colon cells (Figure 4). The BrdU concentration (1 mg / ml) used in drinking water in this study is a concentration generally used for long-term administration without causing any detectable toxicity or weight reduction. It has been reported that drinking water containing 0.8 mg / ml BrdU can be administered to B6 and BALB / C mice for up to 5 weeks without causing thymic toxicity, and drinking water containing 1 mg / ml BrdU is administered to LEW rats for up to 12 weeks. Replacing tissue does not cause significant toxic effects. Inhibitory effects of BrdU on cell proliferation and differentiation in many cell types have been reported in many concentrations in vitro and in vivo. Because BrdU toxicity is species- and cell-type-specific, we cannot fully assess the possible toxic effects from the proliferation of Brdu in LRCs. However, because Lrc continued to divide during de-labeling, based on 2H incorporation of DNa measured by Brdu cells (Table 1) and BrdU-treated rats in our preliminary study showed the same CEC proliferation rate as control rats ( (Data not shown), we conclude that the toxic effect is minimal. The discovery of BrdU cells after prolonged delabeling supports the presence of lRC in the colonic glandular fossa (Figure 5A). The percentage of LRC after the 2-8 week de-labelling period (Figure 6) is 3.8-12%, which is within the previously reported range of values. After screening and collecting the presence and absence of BrdU nuclei, the collected nuclei were screened separately to confirm the remaining differences in fluorescence intensity. The presence and absence of Brdu nuclei form peaks in different fluorescent regions and do not overlap (Figure 5B). Measurements of 2H increase in collected LRCs allowed us to calculate proliferation rates. At 2 ′ 4 and 8 weeks, the 20 mark period (Brdu de-mark period) is based on & merge points 84752 -45- 200401826. There are 11 · 9 ± 8, I4 · 9 soil 6 and I6 · 9 ± 8% new. Synthetic cells. Using the index formula, an average of 0.5% of all LRC populations are divided each day. The accuracy of this method is also supported by studies performed only with long-term administration of 2H2O. Deuterated water alone can be used to determine the presence of LRCs, although it cannot do the separation of LRCs. Rats born after being labeled in the womb (mother had been given 2H2O in drinking water before pregnancy) should have all CECs labeled with 2H, including LRCs and stem cells. Previously in our laboratory, we found that it took less than 1 week to mark to make 2 out of 0 almost completely wash out body moisture. Therefore, cells remaining 2H after 2 weeks can be regarded as LRCs. The percentage of cells that remained 2H after 2 weeks of de-labeling was approximately 10% (Figure 7), which is in good agreement with the results obtained by de-labeling at 2 to 5: 1; We have demonstrated by immunohistochemistry that CEC is effectively labeled by BrdU drinking and drinking. At the same time, the location of wLRC was identified on the unlabeled glandular fossa by immunohistochemistry. The LRC is located at the base of the glandular fossa, which is consistent with the position of colonic stem cells identified by 3 [H] _dT. Digestive gland endocrine cells are known to contain less than <% of total CECs (28). We examined the existence of the population (which has been reported to have a longer lifespan (35-10 () days) than the positively differentiated cecs. Using philophilic staining, we were able to determine the origin of the endocrine cells from the digestive glands in LRC. May be cold-stained to a minimum (Figure 9). This safe in-vivo method using D20 can not only be applied to humans to measure colon epithelial cell proliferation without toxicity, but also can be used to determine the proliferation rate of mature stem cells. For the purpose of understanding slag ^, freshness / eyes, and detailed descriptions with illustrations and examples, for those who are too dying ..., 'U the skilled person should easily take into account the teachings of the present invention 84752-46- 200401826 can do Certain changes and modifications without departing from the spirit and scope of the scope of the attached patent application. The applicant does not waive or contribute to the public any unpatented subject matter related to this article. References 1. American Cancer Society (ACS). (2002) Cancer facts and figures: Colorectal Cancer and Early detection. 2. Wong? WM and Wright, NA (1999). Cell proliferation in gastrointestinal mucosa. J Clin Pathol., 52? 321-333. 3. Risio, M., Coverlizza, S., Ferrari, A., Candelaresi, GL., And Rossini, FP (1998). Immunohistochemical study of epithelial cell proliferation in hyperplastic polyps, adenomas, and adenocarcinomas of the large bowel. Gastroenterology, 94, 899-906 4. Terpstra, OT, van Blankenstein, M ”Dees, J. and Eilers, GA (1987). Abnormal pattern of cell proliferation in the entire colonic mucosa of patients with colon adenoma or Cancer. Gastroenterology, 92, 704-708. 5. Morris, RJ (2000). Keratinocyte stem cells: targets for cutaneous carcinogens. J Clin Invest 106, 3-8. 6. Bach, S. P., Renehan, A. G., and Potten, C. S. (2000). Stem cells: the intestinal stem cell as a paradigm. Carcinogenesis 21, 469-476. 7. Fearon, ER and B. Vogelstein, A. Genetic Model for 84752 -47 -200401826

Colorectal Tumorigenesis. Cell, 61: 759-768, 1990. 8. Pozharisski, K. M., Klimashevski, VF, and Gushchin, V. A. (1980). Study of kinetics of epithelial cell populations in normal tissues of the rat ? s intestines and in carcinogenesis. I. A comparison of enterocyte population kinetics in different segments of the small intestine and colon. Exp Pathol (Jena) 18, 387-406.

9. Bickenbach, JR (1981). Identification and behavior of label-retaining cells in oral mucosa and skin. J Dent Res 60 Spec No C? 1611-1620. 10. Cotsarelis, G., Sun, T. T., and Lavker, RM (1990). Label -retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 61, 1329-1337. 11. Morris, R. J., Fischer , S. M., and Slaga, T. J. (1986).

Evidence that a slowly cycling subpopulation of adult murine epidermal cells retains carcinogen. Cancer Res 46, 3061-3066. 12 · Morris, R. J_, Coulter, K ·, Tryson, K ·, and Steinberg, SR (1997). Evidence that cutaneous carcinogen-initiated epithelial cells from mice are quiescent rather than actively cycling. Cancer Res 57, 3436-3443. 13. Lipkin, M., Bell, B.? and Sherlock, P. (1963). Cell proliferation kinetics in the gastrointestinal tract of man. I. Cell 84752 -48- 200401826 renewal in colon and rectum. J. Clin Investigation 42, 767-776. 14. Sunter, JP3 Wright, NA? and Appleton, DR (1978). Cell population kinetics in the epithelium of the colon of the male rat. Virchows Arch B Cell Pathol 26 ^ 275-87. 15. Potten, C.S., Li, Y. Q., O'Connor, P. J., and Winton, D . J ·

(1992). A possible explanation for the differential cancer incidence in the intestine, based on distribution of the cytotoxic effects of carcinogens in the murine large bowel. Carcinogenesis 13, 2305-2312. 16. Yatabe, Y ·, Tavare, S ·, and Shibata, D. (2001). Investigating stem cells in human colon by using methylation patterns. Proc Natl Acad Sci USA 98, 10839-10844. 17. Potten, C · S ·, Booth, C ·, and Pritchard, DM ( 1997). The intestinal epithelial stem cell: the mucosal governor, Int J Exp Pathol 78, 219-243.

18. Macallan, D. C., Fullerton, C. A., Neese, RA, Haddock, K.? Park, SS5 and Hellerstein, M. K. (1998). Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans. Proc Natl Acad Sci USA, 95? 708-713. 19 · Neese, RA, Siler, S. Q ·, Cesar, D ·, Antelo, F · , Lee, D., Misell, L., Patel, K., Tehrani, S., Shah, P., and Hellerstein, M. K. (2001). Advances in the stable isotope-mass 84752 -49- 200401826 spectrometric measurement of DNA synthesis and cell proliferation. Anal Biochem, 298? 198-195. 20 · Neese, RA ·, Misell, L ·, Turner, S ·, Antelo, F., Kim, J., Cesar, D ”Hoh, R., Chu A., Strawford, A., Christiansen, M.? And Hellerstein, MK (2002) Measurement in vivo of proliferation rates of slow turnover cells by H20 labeling of the deoxyribose moiety of DNA. PNAS. 2002; 99: 15345-15350.

21. Kim, J, Neese, RA, Isnard, P, McCune, M and Hellerstein, MK. A method to isolate long-term label retaining cells (putative stem cells) from rat colon of rats. (2002). Proceedings of American Association for Cancer Research, 127. 22. Brasitus, TA5 Isolation of proliferative epithelial cells from the rat cecum and proximal colon. (1982). Anal Biochem, 123 (2), 364-372.

23. Brasitus, TA and RS Keresztes, Glycoprotein metabolism in rat colonic epithelial cell populations with different proliferative activities. (1983). Differentiation, 24 (3), 239-44. 24. Todd, D., Singh, A. J. Greiner, D.L., Mordes, J.P., Rossini, AA, and Bortell, R. A new isolation method for rat intraepithelial lymphocytes. (1999). J Immunol Methods, 224, 111-127 · 84752 -50 -200401826 25. Coles, MC and Raulet, D. Η. NK1.1 + T cells in the liver arise in the thymus and are selected by interactions with class I molecules on CD4 + CD8 + cells. (2000). J Immunol, 164, 2412-2418. 26. Murrill, W. B., Brown, N. M., Zhang, J. X., Manzolillo, P. A., Barnes, S., and Lamartiniere, CA Prepubertal genistein exposure suppresses mammary cancer and enhances gland differentiation in rats. Carcinogenesis, 17: 1451-1457, 1996. 27. Sheehan, D.? And Hrapchak, B. (1980) Theory and practice of Histotechnology (Detroit, MI): Battelle Press, pp. 276-277. 28. Roth, K. A., and Gordon, JI (1990). Spatial differentiation of the intestinal epithelium: analysis of enteroendocrine cells containing immunoreactive serotonin, secretin, and substance P in normal and transgenic mice. Proc Natl Acad Sci USA 87, 6408-6412. 29. Deschner, EE? and Lipkin, M. (1966). An autoradiographic study of the renewal of argentaffin cells in human rectal mucosa. Exp Cell Res 43, 661-665. 30. Kim5 KM? and Shibata D. (2002). Methylation reveals a niche: stem cell succession in human colon crypts. Oncogene 27, 5441-5449. 31. Bjerknes, M. (1996). Expansion of mutant stem cell 84752 -51- 200401826 populations in the human colon. J Theor Biol 178, 38 1-385. 32. Jecker, P., Beuleke, A., Dressendorfer, I., Pabst, R., and Westermann, J. (1997). Long-term oral application of 5-bromo-2-deoxyuridine does not reliably label proliferating immune cells in the LEW rat. J Histochem Cytochem 45, 393-401.

33. Tough, DF, and Sprent, J. (1994). Turnover of naive and memory-phenotype T cells. J Exp Med 179, 1127-1135. 34. Goz? B. (1978). The effects of incorporation of 5 -halogenated deoxyuridines into the DNA of eukaryotic cells. Pharmacol

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35. Rocha, B., Penit, C., Baron, C., Vasseur, F., Dautigny, N., and Freitas, AA (1990). Accumulation of bromodeox-yuridine-labeled cells in central and peripheral lymphoid organs: minimal estimates of production and turnover rates of mature lymphocytes. Eur J Immunol 20, 1697-1708. 36. Boman, B. M., Fields, J. Z., Bonham-Carter, 0, and Runquist, 〇.A. (2001). Computer modeling implicates stem cell overproduction in colon cancer initiation. Cancer

Res 61, 8408-8411. 37 · Zhang T, Otevrel T, Gao Z, Ehrlich SM, Fields JZ, Boman, BM. Evidence that APC requlates surviving expression: a possible mechanism contributing to the 84752 -52- 200401826 stem cell origin of colon cancer. Cancer Res 2001: 61: 8664-8667. Although the foregoing invention has been described in detail with illustrations and examples for the purpose of clarity, those skilled in the art should easily make certain changes and modifications in light of the teachings of the invention without departing from the spirit and scope of the appended patent application. [Brief description of the drawings] The present invention will be better understood by referring to the following drawings. Figure 1 depicts the experimental design of a dual labeled tissue or individual. Figure 2 depicts the incorporation of deuterium into DNA's deoxyribose (dR) from water. Figure 3 depicts the DNA-labeled pathway in individual cells. Abbreviations used: G: glucose; GNG: gluconeogenesis; P: phosphate; R: ribose; DNPS: purine nascent synthesis pathway; DNNS: nucleic acid nascent synthesis pathway; NDP: nucleoside diphosphate; RR ·· ribonucleoside Reductases; dNTPs · deoxyribonucleoside triphosphates; DNA: deoxyribonucleic acid; dN: deoxyribonucleosides; dT: thymine deoxyribonucleosides; BrdU: brominated deoxyuracil nucleosides. Figure 4 depicts the increase in body moisture during BrdU de-labeling / 2H2O labeling. Figure 5 depicts the nuclear distribution of BrdU-administered rats by FACS analysis. Nucleus profiles from colonic epithelial cells with and without BrdU are shown. A: The nuclei of BrdU rats were intraperitoneally injected. B: Nuclei of BrdU rats administered orally. BrdU-free regions were established by staining colon epithelial cells (CECs) without BrdU treatment with isotype control antibodies. Note that rats injected intraperitoneally have higher fluorescence than BrdU nuclei in rats treated orally. Because toxicological effects were observed by intraperitoneal injection of 84752 -53- 200401826, we decided to use oral. Figure 6 depicts the distribution of CECs de-labeled by BrdU. Nuclei of CECs were obtained from rats delabeled with BrdU at 2, 4, and 8 weeks and stained with anti-BrdU FITC antibody. A BrdU-free control group was used to establish a BrdU-free region. A. 1) The distribution of BrdU-labeled rat nuclei from 2 weeks. 2) From 4 weeks BrdU went to know the distribution map of the nucleus of the white rat. 3) Nucleus distribution of BrdU-labeled rats from 8 weeks. Because LRCs proliferate slowly during BrdU labeling, their fluorescence is not as bright as that of normal differentiated CEC (Figure 4). B. The presence and absence of BrdU nuclei screened by FACS was reanalyzed to confirm that there was no duplication between the two populations. Figure 7 depicts the average LRC ratios of CECs de-labeled by BrdU at 0, 2, 4 and 8 weeks (77 ± 12, 12 ± 11, 7 ± 6.3 and 3 · 8 ± 1%, respectively). We can detect the presence of low but recognizable LRCs and further collect them for GC / MS analysis for deuterium increase. Figure 8 depicts the percentage of LRC measured by de-labeling 2H2O. The filled bars show an increase in deuterium from CEC in labeled uterine rats (n = 4). Blank bars show increased deuterium (ny) in CEC from rats that were unlabeled at 2 weeks. Figure 9 depicts immunohistochemistry of BrdU cells from fully BrdU-labeled and 4-week-delabeled glands. A) BrdU cells were present on the fully BrdU labeled glandular fossa (2 weeks on BrdU). Note the uniform staining (integrated dots) of anti-BrdU antibodies on the cells along the inner glandular fossa. B) BrdU cells were present in the gland follicles marked by BrdU at 4 weeks. Note the location of the LRCs near the bottom of the glandular fossa (boxed out). Figure 10 depicts the angiotropic staining of digestive gland endocrine cells in the colonic gland fossa. Of the 33 stained slides, we can only find digestive glands -54- 84752 200401826 endocrine cells in only 2 slides. Furthermore, its location does not match the known location of LRCs at the base of the glandular fossa. Therefore, LRC contamination by digestive gland endocrine cells appears to be minimal. 55- 84752

Claims (1)

200401826 Scope of patent application: A method for self-organizing or individual separating human, person, soil, soil, soil, or multiple stem cells. The method includes: a) one or more cell lineage notes marked to the Tissue or individual-4 and sufficient time for the-武 夕 # 4 evening cell lineage annotation mark to be incorporated into the cells of the tissue or individual; b) interrupt the administration step a)-a period of time sufficient to form the first of the cell The first group of the narrow group and the field group, where the second group of cells is better than the first group of cells Donggu &amp; 々f. There are more detectable cell lineage annotation marks; and c) detecting the presence or absence of the one or more cell lineage annotation marks in the first population of cells and the second population of cells; and d) the separation of cells The cells of the second group, wherein the cells of the two groups of cells are stem cells. 2. The method according to the scope of the patent application, wherein the first group of cells does not contain detectable and detectable cell lineage annotation marks. 3. The method according to item i of the patent application, wherein the cell lineage noted that the root S has been tritiated DNA. Γ, (The method according to item 3 of the scope of patent application, wherein the toothed DNA is brominated deoxyuracil core: y: or iodized deoxyuracil nucleoside. Human 5. Method, wherein the tissue is selected from the group consisting of the intestine, breast, small intestine, cervix, prostate, skin, bone marrow, liver-heart, osteoblast, thymus, thyroid, pancreas, bladder, lung, biliary technique, nest, testis The group consisting of brain, brain, and lymphoid tissues. E 84752 200401826 6. The method according to item 1 of the scope of patent application, wherein the cell lineage annotation marker is detected by an antibody specific to the cell lineage annotation marker. 7. According to The method of claim 1 in the patent scope, wherein the separation step (c) includes i-fluorescence-activated cell sorting (FACS). 〇8. A method for identifying carcinogenic effects in tissues or individuals, the method It includes: a) isolating one or more stem cells according to the method of the first patent application scope; and b) detecting the DNA modification in the one or more stem cells, wherein the DNA modification is related to carcinogenesis turn off. 9. The method according to item 8 of the application, wherein the DNA modification is selected from the group consisting of chemical modification of DNA, cross-linking, DNA mutation, base deletion, base insertion, and insertion. 10. The method according to item 8 of the scope of patent application, further comprising correlating the ONA modification to a risk factor selected from the group consisting of carcinogen exposure, DNA repair ability, oxidative damage, and mutation risk. 1 1. A method of forging foot chemical reagents to be genotoxic, the method comprising the steps of: a) tissues or individuals administered to the chemical reagents; b) isolating one or more stem cells according to the method of the scope of patent application item 1; and c) DNA modification detected in the stem cell, wherein the DNA modification in the M stem cell identifies the chemical reagent as genotoxicity 0 84752 200401826 Gen Yushen's method of patent scope item 11, wherein the DNA modification is It is selected from the group consisting of DNA damage, DNA mutation & chemical change of DNA. 13. A method for labeling one or more phenotypes of M-derived stem cells, the method comprising: a) isolating one or more stem cells in accordance with the method of the patent application item 丨; and, b) one or more than Che Yiyi Stem cell phenotype and one or more non-stem cell phenotypes to identify the phenotypic difference between the one or more stem cells and the one or more non-stem cells, where the difference is a phenotypic marker of the one or more stem cells. 14. A method for determining the proliferation rate of one or more stem cells in a tissue or an individual, the method comprising: labeling one or more cell lineage annotations to the tissue or individual for a period of time without allowing the cell lineage Annotation markers are incorporated into the tissue or individual; 'b) administration of one or more isotopically labeled DNA synthesis precursors to the tissue or individual; c) interrupting the administration step a) for a sufficient time to form the first population of cells And the second population of cells, wherein the second population of cells contains more detectable markers of cell lineage than the first population of cells; d) the cells detected in the first population of grandchildren and The presence or absence of the one or more cell lineage annotation markers in the cells of the second group; e) isolating the cells of the second group, wherein the cells of the second group 84752 200401826 cells are stem cells; and I) testing The isotope of ribonucleosides that are more or less dilute than the scales of the cheeks is determined by the tissue or individual-the rate of Toxic A. The rate of proliferation of r or more stem cells 〇 The first of the cells 15. The method according to item 14 of the scope of the patent application does not contain a detectable marker. The method according to item 14 of the scope of the patent application, wherein the one or more isotope-labeled flooding precursors is selected from the group consisting of 3H_dT,% glucose, and H20. 17. The method according to item 14 of the scope of patent application, wherein the measuring step e) comprises measuring the hydrolysate of the-or more deoxyribose nuclear #. According to the method of claim 17 of the patent, the one or more hydrolysates are chemically modified. 19. The method according to item 14 of the scope of patent application, wherein the measurement step includes detection by: ¾ free mass spectrometer, liquid scintillation counting, gamma counting, and analysis method of a group consisting of nuclear magnetic resonance and vibratory chirping The isotope of the one or more deoxyribonucleosides is increased. 20. According to the method of the 19th patent application park, the analysis method in # is a mass spectrometer. The method according to claim 14 of the patent scope, wherein the tissue is selected from the group consisting of: , Lung, bile duct, 2 ^ testis, and lymphoid tissue. 2. The method according to item 14 of the scope of the patent application, further comprising a population expansion factor for calculating the first group of cells in the 84752 200401826. 23. · A method for identifying a therapeutic compound, comprising: administering the compound to a first group of one or more tissues or individuals · ^ determining the number of one or more electricity or individuals according to the method of item 14 of the scope of patent application -Proliferation rate of stem cells or multiple stem cells: = According to the method of patent application No. 14 in the patent, the proliferation rate of mosquitoes or multiple tissues in both groups-or stem cell proliferation rates, wherein the compound has not The two ethnic groups administered to the one or more tissues or individuals; and d) comparing the proliferation rate of the one or more stem cells of the first group and the proliferation of the one or more stem cells of the first group Rate, the difference between the proliferation rate of the one or more stem cells of the first group of Zhongrong and the proliferation rate of the one or more stem cells of the first group is sufficient to make the compound a therapeutic compound. 24. The method according to item 23 of the scope of patent application, wherein the compound is a chemical compound, a dietary factor, or a chemical protective agent. • A method according to item 23 of the patent application, wherein the compound is a dietary factor. • The method according to item 25 of the patent application, wherein the dietary factor is a group consisting of free soybean-derived products, rapeseed-derived products, and antioxidants. 27. The method according to item 25 of the scope of patent application, wherein the dietary factor is a soybean-derived product selected from the group consisting of genistein and lunasin. 84752 200401826 A method for demonstrating a compound that stimulates stem cell proliferation, the method comprising: a) the first group of the compound to one or more tissues or individuals; father): determined according to the method of the scope of patent application No. 14-or more Proliferation rate of one or more stem cells from one of the first group of three tissues / solid tissues: :) Determine one or more stem cells from the first group of one or more tissues according to the method of item 14 of the patent application scope The proliferation rate, in which tritium is not administered to the one or more tissues or individuals of a group of dreams; and X-d) comparing the proliferation rate and salty rate of the one or more stem cells of the first group -The proliferation rate of the one or more stem cells of each group, wherein the proliferation rate of the one or more stem cells of the first group is higher than the proliferation rate of the one or more stem cells of the second group; the compound is identified as Therapeutic compounds. 30. The method according to item 29 of the application, wherein the compound is a presumed lymphocyte synchronizing stimulus and the tissue is one or more T cells. • The method according to item 14 of the scope of patent application, further includes measuring ethnicity or jealousy consumption. 2. A method for identifying a disease or disorder by determining the proliferation rate of one or more stem cells according to the method of the 14th member of the scope of patent application. 0. A method according to item 32 of the scope of patent application, wherein the disease or disorder is diabetes and the tissue contains one or more pancreatic beta cells. 84752 200401826 34. The method according to item 32 of the application, wherein the tissue is one or more T lymphocytes and the disease or disorder is immune damage. 35. The method according to item 32 of the scope of patent application, wherein the immune damage is a progressive lymphatic system disorder. 36. The method according to item 35 of the patent application, wherein the progressive lymphatic system is acquired acquired immune deficiency syndrome (AIDS). 37. The method according to item 32 of the patent application, wherein the tissue comprises a Or multiple bone marrow cells and the disease or disorder is gvhd, graft versus host disease. 38 · A reagent set for isolating one or more stem cells in a tissue or an individual, including: ^ a cell lineage annotation mark, and b) instructions for using the reagent set, wherein the reagent set is used to isolate the tissue or the individual from the One or more stem cells. J 9 · The reagent set according to item 38 of the scope of patent application, further including an apparatus for isolating one or more stem cells from the tissue or individual. 40. The reagent set according to item 38 of the scope of patent application, further comprising a means for administering an annotation mark of the cell lineage. 41 · A reagent set for determining the proliferation rate of one or more stem cells in a tissue or an individual, comprising: a) a cell lineage annotation label, and b) an isotopically labeled DNA synthesis precursor, 84752 200401826 where the reagent group is used to determine a proliferation rate in tissues or individuals. • According to the reagent set of Patent Application No. 4 丨 of the Chinese Patent Garden, one or more stem cells are isolated from each limb. 43. According to the patent application of the Chinese Patent Law, the test agent group, the individual isolates the stem cell or multiple types of cells: 44. According to the method, please mark the cell lineage and mark the tool. 45 · = Π Patent range 41 ~ Agent composition DNA synthesis precursor. 84752 or more stem cells are further contained from the tissue or are further contained for administering the